Use of Botrytis cinerea for obtaining gold nanoparticles

ABSTRACT

The present invention is related to the use of  Botrytis cinerea  strains, its spores, hyphae mycelium, sclerotia, intra and/or extracellular organic molecules, such as proteins, nucleic acids, polysaccharides, lipids and secondary metabolites for the biosynthesis of gold nanoparticles (AuNps). In general terms, the present invention is focused to use  B. cinerea  strains and/or molecules generated by this organism for the biological synthesis of AuNps, being then the field of application, the synthesis of nanomaterials, specifically AuNps using the phytopathogenic fungus  B. cinerea  and/or its intra or extracellular proteins purified individually or in combination thereof or any of other intra and/or extracellular molecule produced by this organism as a biological system of synthesis. The metallic nanoparticles are used in various applications including: semiconductors, photoluminescence, biomedicine, imaging for the medical diagnostic, catalysts (dispersed and supported) and in therapies against some types of neoplasia (cancer), among others.

FIELD OF THE INVENTION

The present invention relates to the use of Botrytis cinerea strains,its spores, hyphae, mycelium and/or sclerotia and/or molecules generatedby this organism, such as proteins, nucleic acids, polysaccharides,lipids and secondary metabolites, for the biosynthesis of goldnanoparticles (AuNps). In general terms, the present invention isfocused to use B. cinerea strains and/or molecules generated by thisorganism for the biological synthesis of AuNps. Therefore, the field ofapplication is centered in the synthesis of nanomaterials, specificallyAuNps using the phytopathogenic fungus B. cinerea and/or its intra orextracellular proteins purified individually or in combination thereofor any of other molecule produced by this organism as a biologicalsystem of synthesis.

The metallic nanoparticles are used in various applications including:semiconductors, photoluminescence, biomedicine, imaging for the medicaldiagnostic, catalysts (dispersed and supported) and in therapies againstsome types of neoplasia (cancer), among others.

BACKGROUND OF THE INVENTION

The nanoparticles are structures with a size ranging from 1 to 100nanometers and are especially attractive due to its optical, chemical,photoelectrochemical and electrical properties (Wilson M., KannangaraK., Smith G, Simmons M., Raguse B. Nanotechnology: Basic Science andEmerging Technologies. Chapman and Hall/CRC 2002; Jain, P. K., Huang,X., El-Sayed, I. H. El-Sayed, M. A. 2008. Noble metals on the nanoscale:optical and photothermal properties and some applications in imaging,sensing, biology and medicine. A of Chem Res. 41:1578-1586).

The synthesis of nanoparticles of different compositions and sizes is afield of investigation of great interest in the last years. Currently,the large scale production of AuNPs is carried out by chemicalprocesses, which require the use of reducing agents to generate theparticles from soluble gold salts. There are also physical processes,which require operating at reduced pressures and high temperatures. Inboth cases associated with AuNPs production, are produced chemical toxiccompounds, due to the reactive agents and the operating conditions ofthe signaled systems; which present problems related to stability,aggregation and control of the desired size of the generatednanoparticles (Sau T. K., Murphy C. J. 2004. Room temperature,high-yield synthesis of multiple shapes of gold nanoparticles in aqueoussolution. J Am Chem Soc 126:8648-8649).

Given the relevance of this topic worldwide, indispensable is the needto implement alternative and efficient processes for obtaining metallicnanoparticles that are “environmentally friendly” without requiring highquantities of energy. In this regard, biological systems are goodcandidates to do this. Currently, there are various publications on thistopic, specifically related to the capacity of some organisms togenerate these structures including bacteria and fungi (Brown S,Sarikaya M, Johnson E A. 2000. Genetic analysis of crystal growth. J MolBiol 299: 725-735; Nair B, Pradeep T. 2002. Coalescence of nanoclustersand formation of submicron crystallites assisted by Lactobacillusstrains. Cryst Growth Des 2: 293-298; Husseiny M I, Abd El-Aziz M, BadrY, Mahmoud M A. 2007. Biosynthesis of gold nanoparticles usingPseudomonas aeruginosa. Spectrochimica Acta Part A 67: 1003-1006;Narayanan K B, Sakthivel N. 2010. Biological synthesis of metalnanoparticles by microbes. Adv Colloid Interface Sci 156: 1-13;Thirumurugan G, Veni V S, Ramachandran S, Rao J V, Dhanaraju M D. 2011.Superior wound healing effect of topically delivered silver nanoparticleformulation using eco-friendly potato plant pathogenic fungus: synthesisand characterization. J Biomed Nanotechnol. 7: 659-66; Mourato A,Gadanho M, Lino A R, Tenreiro R. 2011. Biosynthesis of crystallinesilver and gold nanoparticles by extremophilic yeasts. Bioinorg ChemAppl. 2011: 546074; Balagurunathan R, Radhakrishnan M, Rajendran R B,Velmurugan D. 2011. Biosynthesis of gold nanoparticles by actinomyceteStreptomyces viridogens strain HM10. Indian J Biochem Biophys 48:331-335; Tikariha, S.; Singh, S.; Banerjee, S.; Vidyarthi, A. S. 2012.Biosynthesis of gold nanoparticles, scope and application: A review.IJPSR 3: 1603-1615).

The probable mechanisms by which peptides, bacteria, fungi, and plantscatalyze the extracellular synthesis of metal nanoparticles have beenrecently revised (Durán N, Marcato P D, Durán M, Yadav A, Gade A, Rai M.2011. Mechanistic aspects in the biogenic synthesis of extracellularmetal nanoparticles by peptides, bacteria, fungi, and plants. ApplMicrobiol Biotechnol 90: 1609-1624).

B. cinerea is a phytopathogenic fungus which infects a large number ofvegetal species of great economic importance including fruit trees,ornamental plants and vegetables. This fungus produces a disease knownas grey mold generating a serious problem on pre and postharvest instrawberries, raspberries, apples, pears, chestnuts, kiwi and grapesamong others. In the grapevine, this fungus produces the bunch rot, (vanKan J. A. 2006. Licensed to kill: the lifestyle of a necrotrophic plantpathogen. Trends Plant Sci. 11, 247-253; Elad, Y., Williamson, B.,Tudzynski, P. and Delen, N. eds. 2007. Botrytis: Biology, Pathology andControl. The Netherlands: Kluwer Academic Publishers).

Traditionally, B. cinerea has been studied with the objective ofgenerating strategies to allow its control, and thus, to reduce theeconomic loss associated with the infections generated by the fungus. Upto today, there are no studies in nanotechnology field where cultures,propagules or molecules of B. cinerea are used in the process ofsynthesis of metallic nanoparticles. Our results show that B. cinerea inliquid medium is able to catalyze the synthesis of gold nanoparticles atroom temperature from a solution of HAuCl₄. The formation ofnanoparticles is verified by the change of color of the reactionsolution from pale yellow to reddish o purple. Moreover, the solutionscontaining the nanoparticles present a maximum of absortion at 540 nm,characteristic of the presence of this type of structures (Castro M E,Bravo M, Castillo A. 2012. Biosíntesis de nanopartículas de plata y oropor el hongo fitopatógeno Botrytis cinerea. XXI Congreso Latinoamericanode Microbiologia. Santos, Brasil. 28 de Octubre-1 de Noviembre).

Respect to the intellectual property, the patents related to synthesisof metallic nanoparticles mostly consist of the use of chemicalprocesses for the synthesis of these structures, some of them allow theproduction of particles of a certain size and morphology. This is thecase of the U.S. Pat. No. 6,929,675 in which is described a chemicalsystem for the generation of copper, silver and gold nanoparticles. Inspecific relation with the AuNps, it is also possible to find somepublications, like the patent US 20070125196, in which is disclosed thesynthesis of AuNPs in a size ranging from 30 to 90 nm using a aqueousmedium containing sodium acrylate and also the publication US20060021468 in which is described a chemical process to control theuniformity of the generated particles.

Finally, it should be noted that although there are patents related tothe use of biological systems for the synthesis of AuNps, there arecurrently no patents describing the use of B. cinerea or the moleculesproduced by said fungus for such purposes. In this context, the patentof greatest similarity is the patent published by a researcher ofUniversity of Illinois in the year 2010 (Publication US No.20100055199), in which is described the use of the fungus Trichodermareesei for the synthesis of AuNps.

SUMMARY OF THE INVENTION

The present invention corresponds to the synthesis of AuNps mediated bythe filamentous ascomycete B. cinerea and/or molecules secreted by thefungal mycelium. Because of the above, the process can be carried outusing directly the fungus or its molecules in an isolated manner.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Synthesis of extracellular AuNps by B. cinerea. B. cinereaculture supernatant incubated with HAuCl₄ in different wells of ELISAmicroplates. (a) Culture medium without inoculation, (b) culture mediumobtained from cultures of B. cinerea, (1, 5 and 10) culture mediumobtained from cultures of B. cinerea in presence of HAuCl₄ 1, 5 and 10mM, respectively.

FIG. 2. Absorption spectrum of the AuNps generated by B. cinerea culturesupernatants. A maximum of absorption is observed at 550 nmapproximately.

FIG. 3. EDS Spectrum of the gold particles generated by B. cinerea.Signals corresponding to the presence of the gold element as aconstituent of the nanoparticles are observed.

FIG. 4. Transmission electron microscopy of the gold particles generatedby B. cinerea. A great diversity of sizes and morphologies of particlesis observed. The bar at the bottom right corresponds to 100 nm.

DESCRIPTION OF A PREFERRED EMBODIMENT OF THIS INVENTION

Process specifically designed to produce gold nanoparticles fromfilamentous fungi. This process comprises the following steps:

1. Growth of the fungal mycelium: Is cultivated in 250 mL Erlenmeyerflasks containing 50 mL of a nutritive medium containing between 0.1-1%malt extract and 0.1-1% yeast extract. The fungus was cultivated at 20°C. in darkness, and to do this, the flasks were maintained in a darkroom for 10 days.

2. Generation of metallic nanoparticles: In a 500 mL Erlenmeyer flaskwas collected approximately 100 mL of the supernatant liquid obtainedfrom the growth of the fungus and was incubated with HAuCl₄.3H₂O (finalconcentration 0.5-10 mM). To do this, was added to the supernatant0.5-10 mL of a solution of HAuCl₄.3H₂O 100 mM and the 500 mL Erlenmeyerflask was incubated at a temperature ranging between 25-27° C. for aperiod of time ranging from 0.5 to 12 hours. The particles wereretrieved by low-speed centrifugation (6,000-8,000 rpm) o by spontaneoussedimentation leaving the tubes at rest for at least 1 hour.

The material was characterized by: i) adsorption spectrum in which isobserved a maximum at approximately 550 nm. ii) Transmission electronmicroscopy of the gold particles generated by B. cinerea. A greatdiversity of sizes (between 10-300 nm) as well as morphologies of theparticles (spherical, hexagonal, triangular and polyhedral) is observed.

The invention claimed is:
 1. Process for the biological synthesis ofgold nanoparticles (AuNps) by Botrytis cinerea strains, spores, hyphae,mycelium, sclerotia and/or molecules generated by such microorganism,comprising: a) culturing such Botrytis cinerea strains, spores, hyphae,mycelium or sclerotia in a nutritive medium containing between 0.1-1%malt extract and 0.1-1% yeast extract at 20° C. in darkness for at least10 days; and separating a fungal supernatant from such nutritive medium;and b) generating gold nanoparticles by incubating the fungalsupernatant with HAuCl₄.3H₂O, or alternatively extracting the moleculesfrom the fungal supernatant and incubating them with HAuCl₄.3H₂O, for aperiod of time ranging from 0.5 to 12 hours, at a temperature rangingbetween 25-27° C. and retrieving the nanoparticles using acentrifugation speed of 6,000 to 8,000 rpm or by spontaneoussedimentation after a rest of at least 1 hour.